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EFFECTS OF POLIO VIRUS INFECTION ON
MITOCHONDRIAL FUNCTION
byAnna Koundouris
School of Biomedical and Life Sciences
University of Surrey
United Kingdom
A thesis submitted in accordance with the requirements of the University of Surrey
for the Degree of Doctor of Philosophy
March, 2001
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ACKNOWLEDGEMENTS
Many thanks to my supervisor Dr Mike Carter and co-supervisors Dr George Kass
and Pete Sanders for giving me this opportunity to undertake this study and for their
help and advice.
Special thanks to Margaret Carter for her invaluable help throughout my PhD and to
Angie Boxall for her technical support and friendship.
A ‘special’ thank you to those - they know who they are! - who supported me and put
up with me.
An ‘extra’ special thank you to my fiancé Kosta for his support and love.
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ABSTRACTEnteroviruses have a tropism for muscle cells and have been linked to the
development of CFS. Muscle abnormalities made worse by exercise are one of the
major symptoms in CFS and abnormalities in cellular energy levels suggestive of an
impairment of mitochondrial function have been reported following virus infection.
A mitochondrial disorder precipitated by a virus infection has been suggested as the
cause for the unexplained fatigue in CFS. The work presented in this thesis provides
a potential link between CFS and viral infection by suggesting that the energy
abnormalities in patients diagnosed with CFS may be due to a virus-induced
impairment of the mitochondrial electron transport chain. A novel effect on cellular
respiration caused by poliovirus infection was shown. The effect of poliovirus
infection on mitochondrial function was investigated in COS-1 and T47D
mammalian cells. In both cases a rapid decrease in total cell respiration was
observed, and this was attributed to an inhibition of mitochondrial respiration. In
parallel with the inhibition of mitochondrial respiration, the activity of succinate
dehydrogenase was impaired during poliovirus infection. This shows that poliovirus-
induced inhibition of cellular respiration occurs primarily through inhibition of
electron flow at complex II of the mitochondrial respiratory chain. Infected cells
also showed increased staining with the fluorescent lipophilic cationic mitochondrial
probe tetramethylrhodamine ethyl ester showing that the impairment of respiration
does not lead to a collapse of mitochondrial membrane potential and mitochondrial
permeability transition pore opening. The involvement of the poliovirus non-
structrural proteins 2B and 2BC was also investigated. Expression of 2B, but not
2BC, in COS-1 cells also caused a significant increase in mitochondrial membrane
potential indicating that 2B may he responsible for the increased mitochondrial
membrane potential during poliovirus infection. Additionally, work to generate
antibodies specific for these proteins was initiated in order to investigate their
intracellular localisation.
Ill
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ORIGINAL PUBLICATIONS
The following original publication is based on work presented in this thesis.
Koundouris, A., Kass, E. N. G., Johnson, C. R., Boxall, A., Sanders, P. G. & Carter,
M. J. (2000). Poliovirus induces an early impairement of mitochondrial function by
inhibiting succinate dehydrogenase activity. Biochemical and Biophysical Research
Communications 271, 610-614.
IV
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AA
List of frequently used abbreviations
Antimycin A
bp base pairs
CFS chronic fatigue syndromeCIP Calf intestinal alkaline phosphatasecpe cytopathic effectsDa Dalton/s
DEPC diethylpyrocarhonate
DMSO Dimethyl susphoxide
dNTP deoxy-nucleotide-tri-phosphateDTT dithiothreitol
E.coli Escherichia coli
ECL Enhanced Chemiluminescence
EDTA ethylenediaminetetraacetic acidECS foetal calf serum
CDS gel documentation system
GST glutathione S-transferase
hr hour/s
IPTG isopropyl-p-D- thiogalactopyranoside
kb kilobase
kDa kilo Dalton
KOH potassium hydroxide
1 litre
MGS multicloning site
min minute/s
ml millilitre/s
MCID microcomputer imaging device
M-MLV Moloney murine reverse transcriptase enzyme
MOPS 3-[N-morpholino]propanesulfbnic acid
Mr molecular weight
mtDNA mitochondrial DNANADH nicotinamide adenine dinucleotide
ng nanogram
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nt
ORF
P
PBS
PCA
PCR
PFS
Polio I
PV
RC
RF
RI
Rnase
RT mix
RP-HPLC
RT-PCR
SDS
SDS-PAGE
SE
sec
sscSE
SDH
TBE buffer
TEMED
TMRE
TMPD
X-Gal
IDNA
Pgpi
DMEM
GMEM
P-gal
nucleotide
open reading frame
associated probability
phosphate buffered saline
perchloroacetic acid
polymerase chain reaction
post-viral fatigue syndrome
poliovirus type I
poliovirus
replication complex
replicative form
replicative intermediate
ribonuclease
Reverse transcription mix
reverse phase high performance liquid chromatography
reverse transcriptase polymerase chain reaction
sodium dodecyl phosphate
SDS-polyacrylamide-gel-electrophoresis
standard error
second/s
saline sodium citrate
standard error
succinate dehydrogenase
tris boric EDTA buffer
N,N,N',N'-tetramehtylethylenediamine
tetramethylrhodamine ethyl ester
A, A, A', A ’-tetramethyl-1,4-phenylenediamine
5-bromo-4-chloro-3-indole p-D-galactopyranoside
Lambda DNA
microgram
microlitre
Dulbecco’s modified Eagles's basal medium
Glasgow's modified Eagles's basal medium
P-galactosidase
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X-gal 5-bromo-4-chloro-3-indolyl-y5-D-galactoside
INT /7-iodonitrotetrazolium violet
U units
v ii
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Table o f contents
Acknowledgements ii
Summary iü
Original Publications iv
List of frequently used abbreviations v
Table of contents viii
List of tables xiii
List of figures xiv
CHAPTER 1 : INTRODUCTION 1
1.1 Chronic fatigue syndrome 2
1.1.1 Case definition 3
1.1.2 Clinical epidemiology and presentation 4
1.1.3 Aetiology 6
1.1.3.1 Enteroviruses 7
1.1.3.2 Muscle abnormalities 8
1.2 Human poliovirus 9
1.2.1 Classification 10
1.2.2 Viral pathogenesis 12
1.2.3 Structure 12
1.2.4 Physical and chemical properties 15
1.2.5 Cytopathic effects 15
1.2.6 Membrane alterations 16
1.3 Molecular biology of the human poliovirus 21
1.3.1 TheRNA genome 21
1.3.1.1 Structure 21
1.3.1.2 The polyprotein 23
1.3.2 Replication and the replication complex 26
1.4 Mitochondria 30
1.4.1 Structure 31
1.4.2 Functions of mitochondria 33
1.5 Research objectives 37
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CHAPTER 2:MATERIALS AND METHODS 39
2.1 Materials 40
2.1.1 Suppliers 40
2.1.2 Solutions and buffers 43
2.1.2.1 Microbial growth medium 43
2.1.2.1 Buffers used in molecular cloning 44
2.1.2.3 Agarose gel DNA electrophoresis buffers 45
2.1.2.4 Northern blotting solutions 45
2.1.2.5 SDS-PAGE solutions 47
2.2 Mammalian cell culture 49
2.2.1 Cell lines 49
2.2.2 Freezing of cell lines 50
2.2.3 Cell counting 51
2.3 Viral culture techniques 51
2.3.1 Virus strain and growth 51
2.3.2 Virus titration (plaque assay) 52
2.3.3 Virus infection of cell lines 53
2.4 Measurement of mitochondrial membrane potential with tetramethylrhodamine ethyl
ester 53
2.5 Measurement of oxygen consumption 54
2.5.1 Measurement of total oxygen consumption 54
2.5.2 Measurement of mitochondrial electron chain activity 54
2.6 Assay of succinate dehydrogenase activity 55
2.7 RNA extractions 55
2.8 Cloning Techniques 56
2.8.1 Isolation of plasmid DNA from bacterial transformants 5 6
2.8.1.2 Alkaline lysis-diatomaceous earth method (Carter & Milton, 1993) 56
2.8.1.2 S.N.A.P. Method 58
2.8.1.3 Alkaline lysis / PEG precipitation method 58
2.8.2 Purification of DNA 58
2.8.2.2 Precipitation of PCR products 59
2.8.2.3 Purification of PCR products, DNA plasmids, and fragments 59
2.8.3 Enzymes 60
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2.8.3.1 Restriction enzyme digests 60
2.8.3.2 Production of blunt-ended DNA 60
2.8.3.3 Déphosphorylation of DNA 61
2.8.3.4 DNA ligations 61
2.8.4 Growth and manipulation of bacteria cells 62
2.8.4.1 Preparation of competent E.coli DH5a cells 62
2.8.4.2 Transformation of E.coli DH5a cells with plasmid DNA 62
2.8.5 Agarose gel DNA electrophoresis 63
2.9 Polymerase chain reaction 64
2.9.1 Preparation of oligonucleotide primers 64
2.9.2 Reverse Transcription-PCR amplification of RNA (RT-PCR) 64
2.9.2.1 Reverse transcription of RNA 64
2.9.2.2 PCR amplification 65
2.9.3 PCR amplification of DNA 66
2.10 DNA sequencing 66
2.11 Northern blotting 67
2.11.1 RNA electrophoresis 67
2.11.2 Northern transfer 67
2.11.3 Blot hybridisation 68
2.12 Separation of proteins in SDS-polyacrylamide gels 69
2.12.1 SDS-polyacrylamide-gel-electrophoresis (SDS-PAGE) 69
2.12.2 Kenacid blue staining 70
2.13 Western blotting 71
2.13.1 Protein transfer 71
2.13.2 Antigen detection 71
2.14 Expression of recombinant proteins in Escherichia coli 72
2.14.1 Induction of the fusion proteins 72
2.14.2 Purification and screening of induced fusion proteins 73
2.15 Transfection of mammalian cells 74
2.15.1 Lipofectin mediated transfection of COS-1 cells 74
2.15.2 Transfection efficiency 74
2.16 Reverse phase high performance liquid chromatography of nucleotides and
nucleosides 75
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2.17 Fluorescence microscopy to monitor nuclear changes 78
CHAPTER 3:EFFECT OF POLIOVIRUS INFECTION ON MITOCHONDRIAL FUNCTION 79
3.1 Aims of the study 80
3.2 Effect of PV infection on cellular respiration 80
3.3 Mitochondrial respiration 83
3.4 Succinate dehydrogenase activity of PV infected COS-1 cells 91
3.5 Mitochondrial membrane potential 92
3.6 Adenine nucleotides 96
3.7 Summary 97
CHAPTER 4:EXPRESSI0N OF THE POLIOVIRUS NON-STRUCTURAL PROTEINS 2B AND 2BC 98
4.1 Aims of this study 99
4.2 Cloning of the 2BC and 2B coding sequences 100
4.2.1 Cloning of the 2BC coding sequence 103
4.2.2 Cloning of the 2B coding sequence 107
4.3 Expression of 2BC and 2B genes in mammalian cells 110
4.3.1 Sub-cloning of 2BC and 2B coding sequences into a mammalian expression vector 110
4.3.2 Expression of 2BC and 2B proteins in COS-1 cells 115
4.3.3 Effects of the expression of 2BC and 2B proteins on the mitochondrial membrane
potential 117
4.4 Expression of 2BC and 2B genes in bacterial cells 118
4.4.1 Sub-cloning of 2BC and 2B coding sequences into GST gene fusion vectors 118
4.4.2 Expression of 2BC and 2B proteins in E.coli 123
4.4.3 Cleavage of fusion proteins 128
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4.5 Summary 128
CHAPTER 5: DISCUSSION 129
CHAPTER 6: CONCLUSIONS AND FUTURE WORK 141
6.1 Conclusions 142
6.2 Future work 143
REFERENCES 145
APPENDIX I AI
APPENDIX II All
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List o f tables
Table L I Frequency o f symptoms reported in CFS. 5
Table 1.2 Human enteroviruses. 11
Table 3.1 Metabolic supplements o f complex I and II o f the respiratory
chain used in this study. 85
Table 3.2 Inhibitors o f complex I and II o f the respiratory chain used
in this study. 85
Table 3.3 Effect ofpolio I infection on the activity o f SDH. 92
Table 3.4 Effect o f polio I infection on the concentrations o f ATP, ADP
and AMP. 96
Table 4.1 Sequence o f the three primers, 2BCstart, 2BCend, and 2Bend used
for the cloning o f the genes corresponding to the 2BC
and 2B proteins. 101
Table 4.2 The universal primers M l 3 forward and M l 3 reverse. 105
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List o f figures
Figure 1.1 Electron micrograph o f poliovirus and schematic representation
o f the icosahedral structure ofpoliovirus particles. 14
Figure 1.2 Schematic representation o f IP3 signal pathway. 20
Figure 1.3 Genomic organisation o f poliovirus. 22
Figure 1.4 Processing o f the polyprotein. 24
Figure 1.5 Electron micrograph o f a poliovirus replication complex
(RC) surrounded by virus-induced vesicles (V). 29
Figure 1.6 The general structure o f a mitochondrion 32
Figure 1.7 The respiratory chain showing the flow o f electrons from
NADH to oxygen and the extrusion o f proton by complexes
I, III and IV. 34
Figure 2.1 Separation o f ATP (+), ADP (•), AMP (à.), adenosine (J
and inosine ( 4) by RP-HPLC. 77
Figure 3.1 Antimycin A blocks cellular consumption o f oxygen. 81
Figure 3.2a Cell respiration o f COS-1 cells infected with polio I. 82
Figure 3.2b Cell respiration ofT47D cells infected with polio I. 83
Figure 3.3 The respiratory chain showing the flow o f electrons from
NADH to oxygen. 84
Figure 3.4 Effect o f polio I infection on mitochondrial respiratory chain
in COS-1 cells. 88
Figure 3.5a Effect o f polio I infection on electron transport through
cytochrome c. 89
Figure 3.5b Effect o f polio I infection on electron transport through
cytochrome c. 90
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Figure 3.6 Effect o fpolio I infection on TMRE fluorescence in T47D cells. 94
Figure 3.7 Digitised TMRE fluorescence images o fpolio I infected and
mock-infected T47D cells. 95
Figure 4.1 Diagram o f the pTZ19R vector map and its MCS. 702
Figure 4.2 7.2% agarose gel o f PCR products generated to contain the
2BC coding sequence. 103
Figure 4.3 1.2% agararose gel o f EcoRI digested recombinant
pTZ19R/2BC plasmids. 105
Figure 4.4 Sequence chromatograms o f the pTZ19R/2BC recombinant
clone. 106
Figure 4.5 2%> agarose gel o f PCR products generated to contain the 2B
coding sequence. 107
Figure 4.6 2% agararose gel o f EcoRI digested recombinant
pTZ19R/2B plasmid. 70&
Figure 4.7 Sequence chromatograms o f the pTZ19R/2B recombinant clone. 109
Figure 4.8 Map o f the pUSlOOO vector. 111
Figure 4.9 Restriction maps showing the relative KpnIIpositions. 113
Figure 4.10 1.2% agarose gel o f 2BC/pUS1000 constructs digested with
KpnII. 114
Figure 4.11 1.2%) agarose gel o f 2B/pUS1000 constructs digested with
KpnII. 114
Figure 4.12 COS-1 cells transfected with pCHllO. 115
Figure 4.13 Northern blot o f 2B and 2BC RNA. 116
Figure 4.14 TMRE fluorescence in COS-1 cells expressing 2B and 2BC. 117
Figure 4.15 Maps o f the GSTfusion vectors and their MCS. 119
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Figure 4.16 Restriction maps showing the relative KpnII and Pstllpositions
in thepGEX-4T-2 andpGEX-4T-3 constructs. 121
Figure 4.17 1.2% agarose gel o f 2BC/pGEX-4T-2 constructs digested with
Kpnl and Pstll. 122
Figure 4.18 1.2% agarose gel o f 2B/pGEX-4T-3 constructs digested with
Kpnl and Pstll. 122
Figure 4.19 Kenacid blue stained 10%) SDS-PAGE gel o f the bacterial
lysates containing the pGEX-4T-2 fusion vectors. 124
Figure 4.20 Kenacid blue stained 10%o SDS-PAGE gel o f the bacterial
lysates containing the pGEX-4T-2 fusion vectors. 125
Figure 4.21 Western blot o f the fusion proteins following induction o f the
pGEX-4T-2 vectors. 126
Figure 4.22 Western blot o f the fusion proteins following induction o f the
pGEX-4T-3 vectors. 127
Figure 5.1 Multiple sequence alignment o f Human Bcl-2 with several
viral proteins. 138
Figure 5.2 A schematicrepresentation o f the poliovirus effect on
mitochondria 139
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Chapter 1
INTRODUCTION
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Chapter 1
INTRODUCTION
Enteroviruses have a tropism for muscle cells and have been implicated in the
aetiology of cbronie fatigue syndrome (CFS). Abnormal mitochondria have been
reported in CFS patient muscle biopsies and defects in energy supply have also been
suggested as a possible cause of this syndrome. Amongst several possibilities is the
chance that the disease might be triggered by a virus infection in muscle. This
project set out to investigate the possible effect of enterovirus infection on cell
function, and in particular energy generation.
1.1 Chronic fatigue syndrome
Cbronie fatigue syndrome (CFS) is a chronic condition of uncertain aetiology and
lacking an identified definite pathological abnormality (Bock & Whelan, 1993). The
illness is not associated with pathognomonic physical or laboratory abnormalities
and is not a cause of premature death. Definitive treatments for this illness remain to
be identified. Several synonyms have been used to describe the epidemic and
endemic forms of this disease that can have up to seventy symptoms and signs. The
common synonyms are: post-viral fatigue syndrome (PFS), favoured in Great
Britain, cbronie infectious mononucleosis, favoured in U.S.A., and benign or
epidemic myalgic encephalomyelitis (ME). The epidemic type of CFS has been
reported infrequently since the late 1950s (Holmes et a l, 1987). Synonyms used to
identify the epidemic form include epidemic neuromyaestbenia, Adelaide epidemic.
Royal Free disease, and Icelandic disease indicating outbreaks in various
geographical areas. The synonyms used for the endemic form have been chosen on
the assumption that infective agents may be responsible for the syndrome and they
include myelgic encephalomyelitis, idiopathic chronic fatigue syndrome, chronic
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Chapter 1
infectious mononucleosis, “Yuppie” flu and Epstein-Barr disease (Spracklen, 1988;
Shorter, 1993).
1.1.1 Case definition
Case definitions have been developed by the United States Centers for Disease
Control and by British and Australian investigators (Holmes et a l, 1988; Lloyd et
al., 1990; Sharpe et al., 1991). Developing an operational definition of CFS remains
a problem because the concept of fatigue is unclear. In 1994 a revised case
definition was published (Fukuda et a l, 1994). This definition is as follows:
“ A case of the chronic fatigue syndrome is defined by the presence of the following:
1) clinically evaluated, unexplained, persistent or relapsing chronic fatigue that is of new or
definite onset (has not been lifelong); is not the result of ongoing exertion; is not
substantially alleviated by rest; and results in substantial reduction in previous levels of
occupational, educational, social, or personal activities; and
2) the concurrent occurrence of four or more of the following symptoms, all of which must
have persisted or recurred during 6 or more consecutive months of illness and must not
have predated the fatigue: self-reported impairment in short-term memory or
concentration severe enough to cause substantial reduction in previous levels of
occupational, educational, social, or personal activities; sore throat; tender cervical or
axillary lymph nodes; muscle pain, multijoint pain without joint swelling or redness;
headaches of a new type, pattern, or severity; unreffesbing sleep; and postexertional
malaise lasting more than 24 hours.” (Fukuda et al., 1994).
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The inclusion of psychiatric illnesses in the diagnosis of CFS is controversial
amongst physicians. However, pre-existing or co-existing psychiatric disease does
not exclude a person from inclusion in the CFS diagnostic group if the major criteria
of the disease are met and a formal psychiatric evaluation is performed (Bock &
Whelan, 1993).
1.1.2 Clinical epidemiology and presentation
Epidemiologically CFS occurs both sporadically and epidemically with sudden onset
of multiple cases being reported in the U.S.A., Europe, Australasia, and South Africa
since 1934, but the epidemic form seems to have been rare since the late 1950s
(Acbeson, 1959; Shorter, 1993; Briggs & Levine, 1994). Most CFS patients are 20-
50 years of age; women are affected more commonly than men. Group studies have
shown that patients diagnosed with CFS are predominantly white between the ages
of 20-40 years of age (Sbafran, 1991; Gunn et al., 1993; Komaroff, 1993). However,
some studies have shown that CFS may also present itself in adolescents (Komaroff,
1993; Bell et al., 1994). While there are no ethnic differences, CFS tends to occur in
cooler climates and higher socio-economic groups though this may be an artefact as
suggested by some studies. (Spracklen, 1988; Sbafran, 1991; Gunn et a l, 1993;
Levine, 1994).
Patients are usually physically very active before the onset of illness but afterwards
are unable to perform any physical activity; even the modest physical exertion
produces a striking exacerbation of many of their symptoms and in 30-70% of
patients there is worsening of their fatigue that can last for at least 6 months
(Sbafran, 1991; Bock & Whelan, 1993). Table 1.1 summarises the symptoms
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frequently reported in CFS over the years. The course of illness runs for 2-5 years
but occasionally it may last more than 10 years.
Symptom Frequency (%)Fatigue 100Impaired cognition 50 - 85Depression 50-85Pharyngitis 50-75Anxiety 50-70Post-exertional malaise 50-60Pre-menstrual worsening 50-60Stiffness 50-60Visual blurring 50-60Nausea 50 - 60Muscle weakness 40-70Arthralgias 40 - 50Tachycardia 40-50Headaches 35-85Dizziness 30-50Paraestbesias 30-50Dry eyes 30-40Dry mouth 30-40Diarrhoea 30-40Anorexia 30-40Cough 30-40Finger swelling 30 - 40Night sweats 30-40Painful lymph nodes 30-40Rash 30-40Low-grade fever 20-95Myalgias 20 - 95Sleep disorder 15-90
Table 1.1 Frequency of symptoms reported in CFS. (Taken from Komaroff, 1993)
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Most patients report a gradual recovery with relapses precipitated by over-exertion.
The young and those who receive good medical attention have the best chance of
recovery. The social consequences of the illness can be severe. The patients feel
personally deficient, and alienated and many cut down their social life (Ware, 1993).
1.1.3 Aetiology
The aetiology of CFS is not known but the occurrence of the endemic forms
described above must suggest that in some cases at least the cause may be an
infection. Extensive research has been undertaken to determine whether or not one
or more biological agents cause CFS. However, no agent has been shown to satisfy
the required criteria. Immunologic abnormalities have been found in individual
patients with the syndrome suggesting an association between CFS and altered
immune function (Klimas et a l, 1990; Bucbwald & Komaroff, 1991; Linde et a l,
1992; Vojdani & Lapp, 1999). However, the significance of these abnormalities
remains uncertain since most of them do not appear in the majority of patients, the
heterogeneity within patient groups limits the interpretation of the available assays,
and evidence establishing a link between abnormal immunity (humoral and cellular)
and CFS has yet to be found (Sbafran, 1991; Lloyd et a l, 1993).
Some of the reported associations of the illness, such as the abrupt onset of a fever,
suggest that a viral infection may be the cause. Viruses that have been associated,
but not yet widely accepted or established, with the syndrome include the
enteroviruses, retroviruses, human herpesviruses, and Boma disease virus (Ablasbi et
a l, 2000; Martin, 1997; Takabasbi et a l, 1996; Galbraith et a l, 1995; Cunningham
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et al., 1991; DeFreitas et al., 1991; Cunningham et al., 1990). This research project
addressed the possibility of enterovirus involvement, in particular poliovirus, and
these viruses will be considered in more detail in following section.
1.1.3.1 Enteroviruses
Support for an aetiologic role of enteroviruses in CFS is derived mainly from studies
in the United Kingdom where the syndrome is often referred to as PFS. The
definition of post-viral varies from 24 hours after infection to one month after the
initial episode. However, if the fatigue persists for 6 months or longer then the term
'chronic' is used (Behan et a l, 1985; Arcbard et a l, 1988; Behan & Behan, 1988;
Yousef et a l, 1988; Cunningham et a l, 1990; Gow et a l, 1991; Bowles et a l, 1993;
Galbraith et a l, 1995; Galbraith et a l, 1997). Enteroviruses are the group of viruses
most consistently associated with the syndrome. They have been found in the gut, in
stool and blood samples and in muscle biopsies of CFS patients enforcing the
suggestion that these viruses may be implicated in CFS (Behan et a l, 1985;
Spracklen, 1988; Behan et a l, 1993).
Outbreaks of PFS have often been related temporally to the occurrence of
poliomyelitis and studies of patients have indicated that a virus similar to the
poliomyelitis virus may indeed be involved (Acbeson, 1959; Behan & Behan, 1988;
Shorter, 1993). A condition very similar to CFS and termed post-polio fatigue has
been related clinically, historically or physiologically to poliovirus (PV) infection
(Behan et a l, 1993; Bruno et a l, 1998). Abnormal responses to poliomyelitis
vaccination were observed in patients who bad developed PFS in the outbreak of
Akureyri fever (epidemic neuromyasthenia) which took place in Iceland in the
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Chapter 1
1950s, (Behan et al., 1993). Although no PV was isolated, these responses suggest
indirectly that one was in fact in circulation at that time and that PFS patients may
have already been exposed to such an agent. A recent study carried out on patients
with diagnosed CFS showed that administration of poliovirus vaccine led to altered
immune reactivity and virus clearance in these patients suggesting that they bad
already been exposed to a virus possibly related to polio (Vedbara et al., 1997). That
shared antigenic determinants with the poliomyelitis vaccine virus. However, CFS is
probably not due to the poliomyelitis polioviruses since the number of patients with
CFS has not been reduced since immunisation was introduced, although a related but
distinct virus cannot be excluded.
Coxsackie B viruses have also been implicated with CFS. Chronic shedding of these
viruses has been seen in the faeces of CFS patients, enteroviral RNA sequences have
been found in muscle biopsies and serological studies have shown elevated
neutralising antibodies to these viruses (Bell et al., 1988; Yousef et al., 1988; Behan
et al., 1985; Cunningham et al., 1990; Doesett et al., 1990; Beban et al., 1993;
Galbraith et al., 1997). Search for enteroviruses in muscle of patients diagnosed with
CFS bas shown enteroviral genomic material in the patients' muscles (Arcbard et al.,
1988; Cunningham et al., 1990; Gow et al., 1991; Bowles et al., 1993). However,
none of these findings has proved to be consistent.
1.1.3.2 Muscle abnormalities
Muscle fatigue made worse by exercise is one of the major symptoms in patients
with CFS. Single-fiber electromyography has shown muscle membrane defects in
patients with CFS (Jamal & Hansen, 1989). Nuclear magnetic resonance
spectroscopy has shown that the muscle in some patients diagnosed with CFS
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Chapter 1
undergoes premature intracellular acidosis during exercise and has a prolonged
recovery period indicating dysfunction of respiratory metabolism (Arnold et ah,
1984; Yonge, 1988). Also, the intracellular concentration of ATP has been reported
to be reduced in CFS patients undergoing exercise (Wong et al., 1992).
Additionally, clinical studies have shown that patients with CFS demonstrate reduced
aerobic work capacity (Riley et al., 1990). Elevated serum creatine kinase, a marker
for muscle damage when above normal levels, has also been reported in patients with
CFS (Arnold et al., 1984; Behan et al., 1991). All the above observations are
suggestive of mitochondrial abnormalities in the muscles of CFS patients.
Ultrastructural analysis has revealed morphological abnormalities in muscle
mitochondria consisting of branching and fusion of the cristae, which produce the
appearance of “compartmentalisation” within the mitochondria and increase in
mitochondrial size (Behan et al., 1985; Behan et al., 1991). Furthermore, patients
with CFS have been found to exhibit a deficiency in acylcamitine indicating an
energy metabolism abnormality and a fatty acid metabolic dysfunction in the
mitochondria (Kuratsune et al., 1994). A mitochondrial disorder precipitated by a
virus infection has been suggested as the cause for the unexplained fatigue in CFS
although the data published so far is not conclusive (Behan et al., 1991; Behan et al.,
1993).
1.2 Human poliovirus
Enteroviruses have a tropism for muscle cells and have been implicated in the
aetiology of chronic fatigue syndrome as mentioned above. This project used the
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Chapter 1
Mahoney strain of type 1 PV and examined the effects of replication and virus non-
structural (2B and 2BC) proteins on mitochondria.
PV is the aetiologic agent of poliomyelitis, a human disease affecting the central
nervous system and leading to destruction of motor neurones. An 18* Dynasty
(1580-1350 BC) Egyptian carving is the earliest record of poliomyelitis. It shows a
young man with a withered, shortened leg and his foot is held in a position typical of
flaccid paralysis associated with poliomyelitis (Melnick, 1983). Although PV was
first identified as "’"'poliomyelitis virus"" in 1909, by inoculation of monkeys with
specimens from cases of paralytic poliomyelitis (Melnick, 1983), its importance as a
cause of human disease was not appreciated until 1949 when Enders and his co
workers showed that different strains of PV could be grown in cell cultures (Enders
et al., 1949). This breakthrough in virology led to the development of formalin-
inactivated vaccines by Salk (Salk, 1953) and live-attenuated vaccines by Sabin
(Sabin, 1955). In 1953 the name poliovirus was universally adopted following a
recommendation by the Virus Subcommittee of the International Association of
Microbiological Societies (von Magnus et al., 1955).
1.2.1 Classification
Classification of viruses into families has in the past been mainly based on virion
morphology, the nature of the genomic nucleic acid and replication process. Genera
are defined largely on the basis of physiochemical properties, such as virion density
and acid sensitivity. This is being increasingly superceded by genomic analysis. PV
belongs to the genus Enterovirus of the Picornaviridae, a family of small, non
enveloped, positive-strand RNA viruses. Five other genera are currently included in
10
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Chapter 1
this family of viruses, namely: Cardiovirus, Rhinovirus, Hepatovirus, Aphthovirus
(Santti et al., 1999; Hyypiâ et al., 1997) and Parechoviruses. The addition of three
new genera to classify three viruses that have been given the status of unassigned
species: Equine rhinitis B virus (formerly Equine rhinovirus 2), ‘Aichi-like viruses ’
and Porcine teschovirus (formerly Porcine enterovirus 7), has been proposed by the
Institute for 7\jiimal Health, Pirbright Laboratory, Woking, United Kingdom, and
awaits formal acceptance by the ICTV Executive Committee (King et al., 1999).
Enteroviruses are found in humans and animals. Enteroviruses of animals have
generally been classified by host species, e.g. porcine enteroviruses, bovine
enterovirus and swine vesicular disease virus. Human enteroviruses are subgrouped
into PV, coxsackieviruses, echoviruses and enterovirus types 68-71 (Table 1.2).
Three immunologically distinct serotypes of PV (type 1, type 2 and type 3) have
been recognised that share many biological and structural properties (Toyoda et al.,
1984; Melnick, 1990; Minor et al., 1990).
Serotypes Members
3 Human PV 1,2 ,3
23 Human coxsackie A1-22,24
6 Human coxsackie B 1-6
30 Human echoviruses
4 Human enterovirus types 68-71
Table 1.2 Human enteroviruses.
11
-
Chapter 1
1.2.2 Viral pathogenesis
Our knowledge of how PV produces disease (e.g. poliomyelitis) in a host is limited.
However, the pathologic consequences of acute and limited infection in vivo by PV
are rather easy to follow. PV reaches the central nervous system through the blood
following replication in the lymphoid tissue of the pharynx and gut, including
Peyer’s patches. From the blood the virus infects the meninges lining the spinal
canal and then the anterior horn cells of the spinal cord during the first week of
infection. These cells are destroyed within hours and if enough are affected, the
innervated muscles become paralysed. In the worst cases poliovirus may affect the
brainstem leading to respiratory failure and death. The portal entry of PV is
generally thought to be the alimentary tract via the mouth. Shedding of virus occurs
from the throat and in faeces and thus transmission of infection occurs independently
of invasion of the nervous system, which occurs in only a minority of cases. The
incubation period prior to the onset of the disease varies from 2 to 35 days (Minor et
al., 1990; Oldstone, 1996; Melnick, 1990).
1.2.3 Structure
The precise three-dimensional structure of PV was elucidated by crystallographic
studies (Hogle et al., 1985). It consists of an icosahedral protein shell (5:3:2) (fig.
1.1) with an external diameter of ~300 Â that encapsidates a plus strand of RNA of
roughly 7,500 bases. The virion shell is approximately 2.5 nm thick and has a
relative molecular weight of 8 x 10 ,̂ of which the RNA provides about 32 per cent
and the protein 6 8 per cent by weight. The PV capsid is made up of 60 identical
building units, each containing one copy of each of the four structural proteins, V Pl,
VP2, VP3 and VP4, arranged with icosahedral symmetry. The folding pattern of
12
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Chapter 1
VPl, VP2 and VP3 is similar, resulting in an eight-stranded antiparallel /7-barrel
structure (Wetz & Habermehl, 1982; Hogle et a l, 1985). VP4 lies buried in close
association with the RNA core whereas VPl, VP2 and VP3 are exposed at the
surface of the virion with their N termini located at the interior face of the viral
capsid. However, it has been shown that both VP4 and the N terminus of VPl are
reversibly externalised at 37 °C (Li et a l, 1994). The capsid surface has a corrugated
topography: there is a prominent star-shaped peak at the 5-fold axis of symmetry,
surrounded by a deep depression (the “canyon”), and another protrusion (the
“propeller”) at the 3-fold axis (Filman et a l, 1989).
13
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Chapter 1
a) lOOnm
,VP1
CANYON
VP2 VP3
Figure 1.1 Electron micrograph of poliovirus and schematic representation
of the icosahedral structure of poliovirus particles.
a) Whole vims particles (D antigen approximately 30nm in diameter (taken from
(Minor et al., 1990)). b) Picomavirus particles are icosahedral structures with a
triangular number T=3 packing of VPl, VP2, and VP3 on the surface of the particles
(taken from (Rueckert, 1990)). VP4 is hurried deep inside that particle at the base of
the protomer and is not an integral component of the framework making up the shell.
14
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Chapter 1
1.2.4 Physical and chemical properties
The sedimentation coefficient of intact PV is 155-160S whilst that of the empty
capsid is 70-80S. The buoyant density of the virion in caesium chloride gradients is
1.34 g/cm^ (Mapoles et al., 1978). The low density of the virus implies that the
virion is essentially impermeable. All enteroviruses are acid stable, surviving
exposure to pH 3. This stability probably represents adaptation since these viruses
must pass through the acidic conditions of the stomach in order to gain access to the
gut. Enteroviruses are thermolabile. Their exposure to a temperature of 50 °C
destroys them rapidly. However, in the presence of magnesium chloride, their
inactivation at all environmental temperatures is inhibited (Wallis & Melnick, 1961;
Melnick, 1990). The RNA within the virion is infectious and acts as mRNA for
protein synthesis. Infectivity of the RNA is completely resistant to ribonuclease as
long as the virion remains intact (Rueckert, 1990). A single break in the RNA,
whether free or inside the virus particle, is sufficient to destroy infectivity.
1.2.5 Cytopathic effects
Following virus infection, changes in the morphology of the cells are detectable by
visual and biochemical examination. These changes often referred to as the
cytopathic effects (cpe) of a virus are observed early in PV infection. They possibly
result from the production of viral proteins and nucleic acids, but mainly from
alterations to the biosynthetic capabilities of the infected cells (Lenk & Penman,
1979; Rueckert, 1990; Cann, 1997). The nuclei of the infected cells become
crescent-shaped by 2.5 to 3 hours post-infection and are pushed aside to the cellular
periphery. Chromatin is condensed in clumps that are attached to the nuclear
15
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Chapter 1
membrane. This is accompanied by the appearance of numerous membranous
vesicles in the cytoplasm, beginning first in the vicinity of the nucleus, that continue
to proliferate for many hours until the entire cytoplasm is involved (Dales et a l,
1965). These membranous vesicles are "tear-drop shaped", they are bound by double
lipid bilayers and they have been found to contain markers from throughout the
protein secretory pathway suggesting that they are derived from endoplasmic
reticulum and secretory vesicles (Schlegel et a l, 1996). These vesicles are important
in PV RNA replication as discussed in section 1.3.2. Changes in the permeability of
the plasma membrane, associated with the spreading of the vesicles is also observed
(Carrasco et a l, 1989; Holsey et a l, 1990). Also, the ribosomes become dispersed
and no longer cluster in polyribosomes. Additionally, the appearance of the rough
endoplasmic reticulum is altered. The vesicles bound by rough endoplasmic
reticulum are considerably elongated and are located almost exclusively near the cell
periphery. Leakage of the intracellular components, followed by shriveling of the
entire cell are the cytopathic effects seen during the last stages of infection
(Rueckert, 1990).
1.2.6 Membrane alterations
Changes in the membrane potential of the mitochondria in vitro were observed
during the course of this project. Alterations of the plasma membrane of PV infected
cells may be the cause of cpe and host cell death (Carrasco et a l, 1989). According
to this hypothesis, virus products are targeted to the cell membrane and alter its
function, thereby causing interference with several cellular processes and resulting in
cell lysis and death. It is therefore important to review the membrane alterations
caused by PV.
16
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Chapter 1
Early in PV infection the entry of protein toxins such as a-sarcin is promoted. This
phenomenon has been referred to as “early membrane permeabilisation” and it is
specific to cells that contain the PV receptor (Carrasco, 1981; Almela et a l, 1991;
Carrasco, 1995). Furthermore, this early permeabilisation does not always take place
if the uncoating process of the PV is blocked; the introduction of PV growth
inhibitors such as Ro-09-0410 specifically hinder both the uncoating step of PV and
the early permeabilization to a-sarcin (Almela et a l, 1991).
During PV infection the permeability of cell membranes is profoundly changed
allowing for enhanced permeability to cations and other compounds, a phenomenon
referred to as “late membrane permeabilisation” because it requires viral gene
expression (Carrasco et al., 1989). Additionally, increased passive diffusion
(membrane leakiness) of compounds such as choline, nucleotides, and low molecular
weight antibiotics also takes place (Contreras & Carrasco, 1979; Carrasco et al.,
1989; Irurzun et al., 1993; Carrasco, 1995).
Studies carried out on HeLa cells showed that as early as 2-3 hours post-infection the
Na'^-K’*’ gradient of the cells collapses; the intracellular concentrations of Na^
increase whereas the contents decrease, hence the membrane potential is
disrupted. These changes result partly from the inhibition ofNa^/R"^ ATPase activity
and partly from the increased permeability of the plasma membrane (Nair, 1981;
Schaefer et al., 1982; Lopez-Rivas et al., 1987). The concentrations of and Câ "̂
also change at approximately the same time that monovalent ion concentrations are
affected. It has been shown that PV infection elevates the alkaline intracellular pH
(pHi) and that this increase in pHi promotes viral replication (Holsey et a l, 1990;
17
-
Chapter 1
Holsey & Nair, 1993). However, the underlining mechanism whereby PV raises the
pHi remains unknown though, it has been suggested that it may be due to the
activation of a vacuolar-type (V) proton ATPase involving protein kinase C-
mediated phosphorylation (Perez & Carrasco, 1993; Holsey & Nair, 1993). PV
infection of HeLa cells also leads to an increase in the intracellular calcium
concentration [Ca^^Ji (Irurzun et al., 1993). This increase is coincident with changes
in the membrane permeability to monovalent cations and it reaches a 1 0 -fold by the
fourth hour post- infection. Virus gene expression was found to be necessary for the
increase of [Câ ' Ĵi; introduction of PV genome replication inhibitors (guanidine and
Ro-09-0179) and protein synthesis inhibitors (cycloheximide) block increases in
cytosolic calcium concentration (Irurzun et al., 1993). Recently, it was shown that
the PV protein 2BC is responsible for the increase of cytosolic free calcium
concentrations in HeLa cells infected with recombinant vaccinia viruses expressing
2BC; the expression of 2BC increased [Ca^’̂ Ji in a manner similar to that seen during
PV infection (Aldabe et al., 1997). It is not known whether, 2BC promotes the entry
of extracellular calcium alone, by a direct effect on the plasma membrane, or by an
indirect effect on a cellular protein, such as a calcium channel.
The mechanism by which [Ca^^Ji is increased is not clear. According to Irurzun and
co-workers, the extra calcium comes mainly from the extracellular medium but it is
also possible that some of the increased intracellular calcium may come from the
mobilisation of intracellular stores (Irurzun et al., 1993). Additionally, plasma
membrane pore formation during infection simply allows extracellular calcium ions
to pass through (Carrasco, 1995). Increases in inositol triphosphate (IP3) can also
lead to the release of stored calcium from the endoplasmic reticulum (see below).
18
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Chapter 1
The physical integrity of the membrane phospholipids is also altered during PV
infection. The activity of phospholipase C is enhanced as early as the third hour after
PV infection in HeLa cells (Guinea et a l, 1989; Irurzun et al., 1993). As a result of
this increased activation, choline and phosphorylcholine are released into the
medium and high amounts of IP3 are formed in the cytoplasm of the infected cells.
The increase in IP3 is dependent on the multiplicity of infection used (Guinea et al.,
1989). PV gene expression is required to induce the increase in phospholipase C
activity; inhibitors of PV gene expression, such as guanidine and cycloheximide,
added at the beginning of infection block the choline release. However, not all
phospholipases are enhanced by PV infection since the stimulation of phospholipase
A2 by the calcium ionophore A23187 is hindered soon after PV infection (Guinea et
al., 1989; Irurzun et al., 1993). The repercussions that these modifications of lipase
activity may have for the cellular metabolism of the infected cells and for the
functions of PV are not understood. PV RNA replication has been found to be
physically associated with the phospholipid biosynthesis; the introduction of a
phospholipid-synthesis inhibitor (cerulenin) after virus entry has been shown to
selectively prevent the synthesis of PV proteins when added at the early stages of the
viral protein synthesis (Guinea & Carrasco, 1990). It has been proposed that the
increased intracellular calcium observed in PV infected cells may be due to the high
levels of IP3 as a consequence of its binding to the IP3 receptor located in the
endoplasmic reticulum (fig. 1 .2 ); however this suggestion has not yet been
substantiated (Irurzun et al., 1993; Carrasco, 1995). Nevertheless, the presence of
increased amounts of phospholipids in the plasma membrane destabilises the lipid
bilayer and this may lead to the enhancement of membrane permeability observed in
infected cells.
19
-
Chapter 1
Sensor
ADP ^SERCA ^ V
^ ATPC a^+
Endoplasmicreticulum
Figure 1.2 Schematic representation of IP̂ signal pathway.
The release of IP3 from phosphotidyl inositol 4,5-bisphosphate by phospholipase C
(PLC) through the action of G-protein linked receptor or growth factor receptor
agonists leads to the opening of a selective IPs-regulated channel located in the
endoplasmic reticulum. This in turn causes the rapid discharge of the ER Ca^ ̂pool
into the cytosol followed by the opening of plasma membrane Ca^ ̂ channels to
regulate changes in cytosolic Ca^ ̂ concentration in the form of Ca^ ̂ oscillations
(inset).
Although it has been shown that membrane permeabilisation requires viral gene
expression, little is known about the PV proteins that may be responsible for this
occurrence. The proteins that have been so far associated with membrane
permeabilization are 2B, 2BC, 3A, and 3AB (Lama & Carrasco, 1992; Carrasco,
1995; Doedens & Kirkegaard, 1995; Lama & Carrasco, 1996; Aldabe et al., 1996).
When expressed in E.coli, the PV proteins 2B, 3A and 3AB strongly modify the
20
-
Chapter 1
bacterial cell membrane by increasing its permeability; hygromycin B (a
nonpermeant translation inhibitor) strongly inhibits protein synthesis when bacteria
expressing 2B, 3A or 3AB are exposed to the drug, normal bacteria are not inhibited.
(Lama & Carrasco, 1992; Lama & Carrasco, 1996). Studies in mammalian cells
obtained similar findings. Transient expression of either 2B or 2BC (and to a lesser
extent 3A) in COS-1 and Hela cells caused an increase in the plasma membrane
permeability to hygromycin B (Doedens & Kirkegaard, 1995; Aldabe et a l, 1996).
Expression of these two PV proteins also increased the release of choline and uridine
from preloaded cells (Aldabe et a l, 1996).
1.3 Molecular biology of the human poliovirus
1.3.1 The RNA genome
1.3.1.1 Structure
The PV genome is a single-stranded, positive-sense RNA molecule (messenger-
active) of 7,433^ nucleotides. There is a 5' untranslated region (5' UTR), 740
nucleotides long, which is important in translation and positive-sense RNA synthesis.
This is followed by a single open reading frame, and a short 3' untranslated region (3'
UTR), 72 nucleotides long, which regulates negative-sense RNA synthesis (figure
1.3) (Kitamura et a l, 1981; Racaniello & Baltimore, 1981; Rueckert, 1990).
‘ For the Mahoney strain o f type 1 poliovirus (Racaniello & Baltimore, 1981)
21
-
Chapter 1
5’UTR
Open Reading Frame—
3'UTR
^ — p - | j
-poly (A)
Figure 1.3 Genomic organisation of poliovirus (adapted from (Cuconati et a l,1998)X
The untranslated regions are the most conserved parts of the genome. The 5' UTR
contains a ‘clover-leaf secondary structure known as the 1RES (Internal Ribosome
Entry Site) that is required for initiation of viral protein synthesis (Skinner et a l,
1989; Andino et a l, 1990; Gebhard & Ehrenfeld, 1992; Jacobson et a l, 1993).
Mutations of this region modify the translation of the PV RNA (Svitkin et a l, 1988;
Haller et a l, 1996; Slobodskaya et a l, 1996). The 3' UTR has been less explored. It
has been shown to contain an RNA pseudoknot structure that may be involved in
RNA amplification (Jacobson et a l, 1993). Additionally, a phylogenetically
conserved tRNA-like tertiary structure model for the 3'-terminal folding between all
enteroviruses suggests that the 3' UTR structures are important to the life cycle of
these viruses (Pilipenko et a l, 1992; Mirmomen et a l, 1997). However, polioviruses
with partial deletions of their 3' UTRs have been recovered indicating that the 3'
UTR structures may not be absolute requirements for RNA replication (Todd et a l,
1997; Meredith et a l, 1999). Both ends of the genome are modified, the 5' end by a
covalent attachment of a small, basic protein VPg (virion protein genome) (22 amino
22
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Chapter 1
acids), the 3' end by genome encoded polyadenylation (polyA) (Yogo & Wimmer,
1972; Dorsch-Hasler et a l, 1975; Cann, 1997; Cuconati et a l, 1998). VPg appears
to be necessary for the initiation of PV RNA synthesis and it may also be important
in other stages of PV replication (Morrow & Dasgupta, 1983; Reuer et a l, 1990).
The function of the genetically encoded poly(A) remains unknown.
1.3.1.2 The polyprotein
Translation of the genome leads to the production of a 247 kDa polyprotein which is
proteolytically processed by viral proteases both co- and post-translationally to
generate individual virus proteins (figure 1.4) (Rueckert, 1990; Cann, 1997). The PV
RNA genome contains all of the signals required for translation of the viral
polyprotein and replication of the genome within the cytoplasm. The polyprotein is
initially processed into three precursor polyproteins PI, P2 and P3. The PI precursor
is co-translationally myristoylated and is cleaved into VPO, VP3 and VPl capsid
proteins by the protease 3CD^^°. VPO is then autocatalytically cleaved to the VP2
and VP4 capsid proteins, a step essential for infectivity (Wimmer et a l, 1993). P2
and P3 are the precursors of non-structural proteins involved in the polyprotein
maturation and RNA replication. Proteolytic processing of the P2 precursor yields
three different end products, the cysteine protease 2Â ™, 2B and 2C, and one long-
lived precursor, 2BC (Rueckert, 1990; Wimmer et a l, 1993).
23
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Chapter 1
Open Reading Frame
Translation
Polyprotein
PI P2 P3
lABCD 2ABC
VPO VP3 VPl
VP4 VP2 VP3 VPl
2A 2BC 3AB
3ABCD
3CD
Pro Pol
2B 2C 3A 3C 3D
VPg
Figure 1.4 Processing of the polyprotein (adapted from (Rueckert, 1990)).
catalyses the cleavage of the capsid precursor protein PI away from the nascent
polypeptide at the junction between domains PI and P2 (Toyoda et a l, 1986). 2A^'°
has been found to be involved in the shut-off of cellular mRNA translation, to be a
translational activator of the polyprotein and to be implicated in the RNA replication
(Davies et a l, 1991; Hambidge & Samow, 1992; Wimmer et a l, 1993; Yu et a l,
1995; Lu et al., 1995). The functions and characteristics of 2BC and its cleavage
products 2B and 2C are less well defined. 2BC is processed by 3Ĉ ™ to its cleaved
products 2B and 2C. It has been shown that 2B, 2C and 2BC are contained within
the replicative complex hence suggesting that they have a role in RNA replication
24
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Chapter 1
(Bienz et a l, 1987; Bienz et a l, 1990; Bienz et a l, 1992; Bienz et a l, 1994).
Additionally, 2C and 2BC but not 2B associate with membranes and induce vesicle
formation (Cho et a l, 1994; Aldabe & Carrasco, 1995). It has been demonstrated
that 2C is needed continually for viral RNA synthesis and it may have a function in
determining the virion structure (Li & Baltimore, 1988; Baltera & Tershak, 1989; Li
& Baltimore, 1990). 2C has also been shown to have ATPase and GTPase activities
and to have an affinity for nucleic acids (Rodriguez & Carrasco, 1993; Mirzayan &
Wimmer, 1994; Rodriguez & Carrasco, 1995). Studies on PV 2B mutants have
indicated that 2B may be necessary for RNA amplification and also that 2B
multimerisation, as well as 2BC and 2C multimerisation, is required for the
occurrence of viral replication (Johnson & Samow, 1991; Cuconati et a l, 1998). 2B
has been shown to inhibit the secretion of cellular proteins, and to block the
permeabilisation of the plasma membrane and the disassembly of the Golgi apparatus
(Doedens & Kirkegaard, 1995; Aldabe et a l, 1996; Sandoval & Carrasco, 1997).
The P3 precursor is generated by cleavage at the amino terminus of the 3A-coding
region followed by rapid processing to yield the relatively stable intermediates 3AB
and the protease 3CD^™. Slow processing of the two intermediates generates the four
cleavage end products 3A, the genome-linked protein VPg (also known as 3B), the
protease 3Ĉ °̂ and the RNA-dependent RNA polymerase 3D̂ °̂ (Rueckert, 1990;
Wimmer et a l, 1993). 3AB is a cytoplasmic membrane-associated protein that serves
as a precursor for VPg and 3A (Tagekami et a l, 1983; Richards & Ehrenfeld, 1990;
Lama et a l, 1994). Its role in the PV replication cycle remains largely unknown, but
purified 3AB greatly stimulates the activity of 3D̂ °̂ in vitro (Lama et a l, 1994).
Additionally, 3AB induces cell membrane permeability (Lama & Carrasco, 1996).
25
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Chapter 1
3A is essential for RNA replication and it has been found to inhibit cellular protein
secretion (Doedens & Kirkegaard, 1995; Xiang et al., 1995). VPg is necessary for
the initiation of RNA replication (Takeda et al., 1986; Reuer et al., 1990). 3AB and
to a lesser extent 3B are necessary for the stimulation of the autocleavage of 3CD^™
to 3 C ^ and 3D̂ °* (Molla et a l, 1994). 3Ĉ ™ and its precursor 3CD^™ mediate most
of the cleavages in the polyprotein but not the cleavage of VPO to VP2 and VP4.
Additionally, 3Ĉ ™ irreversibly inhibits cellular translation and transcription (Clark et
al., 1991; Clark et al., 1993; Yalamanchili et al., 1996). 3D̂ °̂ catalyses chain
elongation of viral RNA in virus-infected cells (Flanegan & Van Dyke, 1979; Young
et al., 1985). In addition, the formation of a complex between 3D̂ °̂ and 3AB
stimulates polymerase activity above that of 3D̂ °̂ alone (Paul et al., 1994; Plotch &
Palant, 1995).
1.3.2 Replication and the replication complex
The time required for a complete multiplication cycle, from infection to completion
of virus assembly, ranges from 5 to 10 hours. Approximately 10"̂ -10̂ infectious
virus particles are produced in one infected cell which is finally destroyed allowing
the viruses to be released to infect new target cells (Rueckert, 1990; Hyypia et al.,
1997). To initiate infection the virus is attached to the cellular receptor (CD 155 also
known as PVR), a membrane-anchored glycoprotein (Mendelsohn et al., 1989).
Capsid structural reorganisation is brought about by the interaction with the cellular
receptor(s) leading to exposure of the internal VP4 polypeptide and, subsequently,
uncoating of the RNA genome (Rueckert, 1990; Richards & Ehrenfeld, 1990;
Racaniello, 1996).
26
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Chapter 1
Many details concerning the mechanism of PV replication in vivo remain unknown.
PV RNA replication occurs in the cytoplasm of infected cells by using both newly
translated viral proteins and various host components. The replication occurs in the
replication complex (RC) found associated with smooth membranes and the
cytoskeleton (Caliguiri & Tamm, 1969; Lenk & Penman, 1979; Bienz et a l, 1990;
Rueckert, 1990; Bienz et al., 1994). carries out the replication of the RNA
genome with the aid of other viral and cell host factors. The RNA is replicated in
two steps. First, genomic RNA of the infecting virus is copied into a minus strand,
which leads to the formation of the partially double stranded replicative form (RF)
(Takeda et al., 1986). From the replicative form a partially double stranded
replicative intermediate (RJ) is formed and subsequently progeny RNA strands of
positive polarity are produced (Butterworth et al., 1976; Btchison & Ehrenfeld,
1981). The (+) RNA is then translated and as the concentration of the protein
increases, an increasing fraction of (+) RNA is packaged into virions. Formation of
infective virions is accompanied by a "maturation cleavage" in which most of the
VPO chains are cleaved to form the "mature" four structural proteins VP 1-4
(Rueckert, 1990). Completed virus particles are ultimately released by infection-
mediated disintegration of the host cell.
The RCs are found in the centre of rosettes formed by many virus-induced vesicles
and they remain associated with the outer surfaces of these vesicles (Bienz et al.,
1983; Bienz et al., 1987; Bienz et al., 1992). The RC contains a tightly packed
membrane system itself that encloses the RJ with its nascent (+) RNA (fig. 1.5)
(Bienz et al., 1992). This system interacts with the surrounding virus-induced
vesicles in the last steps of completion of mature progeny (+) RNA to release the
27
-
Chapter 1
completed 36S RNA from the RJ and from the RC to the surface of the rosettes.
Hence, only mature progeny (+) RNA is found on the surface of the rosette, whereas
the RNA-synthesising machinery is enclosed and protected in the interior (Bienz et
aA ,1992).
Recent studies have shown that the rosettes can be reversibly dissociated into their
components, the virus-induced vesicles that are capable of initiation and elongation
of (+) RNA on their own (Egger et al., 1996). Structural analysis showed that the
vesicles have tubular protrusions with parts of the RC attached to them and upon
reassociation, the protrusions extend inwards into the RC in the centre of the rosette.
Immunoprécipitation showed that the vesicles carry sets of nonstuctural (2C, 2BC,
3D, and 3 CD) and capsid proteins (14S pentamers) as well as a RJ that was found to
be attached to the surface of the rosettes. During the course of this study it was also
found that the membranes of the virus-induced vesicles are not necessary for
elongation of viral RNA but are required for initiation of viral (+) RNA synthesis
(Egger er a/., 1996).
The intracellular formation of these vesicles has been attributed to the viral protein
2BC (Bienz et al., 1983; Aldabe & Carrasco, 1995; Cho et al., 1994; Barco &
Carrasco, 1995). The P2 proteins 2B, 2BC, and 2C are contained exclusively within
the RC and the vesicular membranes and expression of the recombinant P2 proteins
2BC and/or 2C in cultured mammalian and yeast cells has confirmed the vesicle-
inducing and membrane-altering properties of these proteins (Bienz et al., 1987;
Aldabe & Carrasco, 1995; Cho et al., 1994; Barco & Carrasco, 1995). However, the
complete role of the P2 genomic proteins in replication is not understood entirely.
28
-
Chapter 1
immgm
Figure 1.5 Electron micrograph of a poliovirus replication complex (RC)
surrounded by virus-induced vesicles (V). Bar, 100 nm. Taken from (Bienz et al,
1992).
29
-
Chapter 1
1.4 Mitochondria
Mitochondria are bacteria-sized organelles, found in the cytoplasm of almost all
types of eukaryotic cells, and their main (but not only) function is the production of
most of the cell’s energy.
Mitochondria are thought to be derived from prokaryotic microorganisms, which
evolved a symbiotic relationship with their eukaryotic hosts (Gray et al., 1999;
Duchen, 1999; Wallace & Starkov, 2000). As a consequence of that origin,
mitochondria still own an autonomously replicating and expressing genome of about
16.6 kb, the mitochondrial DNA (mtDNA). Studies of mtDNA and its expression
have further accredited the eubacterial roots of this genome (Gray & Doolittle, 1982;
Gray et al., 1998). Furthermore, elucidation of different mitochondrial genomes has
enabled scientists to trace the evolutionary ascendants of mitochondria to a single
ancestor, the prokaryote Agrobacterium tumefaciens, related to the a subdivision of
the so-called purple bacteria (also known as Proteobacteria) (Yang et al., 1985). The
present boundaries of the evolutionary divide between mitochondria and their
eubacterial relatives are currently inscribed by the published complete sequences of
the obligate intracellular proteobacterium Rickettsia prowazekii (R. prowazekii) (the
causative of epidemic louse-borne typhus) (Andersson et al., 1998) and the
freshwater protozoon Reclimonas americana {R. americana) (Lang et al., 1997).
Phylogenetic analysis indicates that R. prowazekii is more closely related to
mitochondria than is any other microbe studied so far, thus identifying its genome as
the most mitochondria-like eubacterial one (Andersson et a l, 1998; Yang et al.,
1985; Gray et al., 1989). R. americana mtDNA more closely resembles the ancestral
mitochondrial-like eubacterial genome than any other mtDNA investigated so far, as
30
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Chapter 1
exhibited by the eubacterial characteristics of the structure and expression of its
genome (Lang et a l, 1997).
The R. prowazekii genome sequence has enforced the association to the
mitochondrial genome. However, the search for mitochondrial genomes even more
ancestral than that of R. americana continues in an effort to uncover even larger,
more gene-rich mtDNAs. In addition, mitochondrial protein-coding sequences and
genome data may ultimately aid the elucidation of the phylogenetic relationships that
nuclear gene sequences are currently unable to resolve.
1.4.1 Structure
Mitochondria are intracellular organelles, varying in both shape and size. They may
be spherical or elongated, or even branched with a typical size between 0 .7 -1 pm
(Alberts et ah, 1994; Nicholls & Ferguson, 1997). The abundance of mitochondria
varies among cell types, for example, thymus lymphocytes contain 6 - 1 2 organelles,
whereas typical fibroblasts contain a massive and dynamically fluctuating network
composed of an indefinable number of single interconnected mitochondria (Wallace
& Starkov, 2000).
Regardless of the cell type and despite the wide variety in number and morphology,
all mitochondria share several fundamental structural properties (Alberts et ah, 1994;
Nicholls & Ferguson, 1997; Wallace & Starkov, 2000). The appearance of a section
through a typical mitochondrion is shown in figure 1 .6 .
31
-
Chapter 1
Outer membrane Intermembrane space
Inner membrane
«»»
«• »
1
»•
## •# •« •»»
cristaeATP synthase
Figure 1.6 The general structure of a mitochondrion. Taken from (Nicholls &
Ferguson, 1997).
Each mitochondrion is bound by two highly specialised lipid bilayer membranes.
Together they create two separate mitochondrial compartments: the internal water-
containing matrix space and a much narrower intermembrane space. The matrix
contains hundreds of enzymes including those of the tricarboxylic acid cycle and the
(3-oxidation pathway. It also contains several copies of the mtDNA genome, transfer
RNAs, special mitochondrial ribosomes and various enzymes required for expression
of the mitochondrial genes. The outer membrane is rich in cholesterol and contains
several proteins termed porins, which act as non-specific pores for ions and solutes
up to 14 kDa. It also contains embedded or attached enzymes that interface the
mitochondrion with the rest of the cellular metabolic network. The inner membrane
is folded into numerous cristae thus increasing its total surface area. It is not freely
permeable to metabolites and ions but it contains specific transport proteins, which
regulate the passage of selected metabolites into and out of the matrix. It also
32
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Chapter 1
contains proteins that carry out the oxidation reactions of the respiratory chain and
the enzyme complex ATP synthase, which makes ATP in the matrix.
1.4.2 Functions of mitochondria
Mitochondria are primarily ATP generators and the principal form of energy
generated in mitochondria is the so-called electrochemical proton gradient that is
produced by the three of the four respiratory enzyme complexes of the mitochondrial
respiratory chain (or electron transport chain), found on the inner membrane of the
mitochondria (fig 1.7) (Mitchell & Moyle, 1967; Nicholls & Ferguson, 1997). This
gradient supplies the energy required to produce ATP, and to support other activities
of the mitochondria, such as the electrophoretic or protonophoric transport of ions,
metabolic substrates, and proteins destined for the mitochondrial matrix. ATP is
required to drive the majority of energy-requiring reactions such as phosphorylation
reactions that modulate a number of essential cellular processes, it may be stored as a
(neuro)transmitter, and it controls the activity of several classes of ion channel
including the ATP-sensitive channel, the calcium release channel of sarcoplasmic
reticulum and voltage-gated calcium channels (Duchen, 1999).
The mitochondrial respiratory chain comprises a series of reduction/oxidation
reactions within complexes I, II, III, and IV (fig. 1.7). These reactions are linked by
ubiquinone and cytochrome c (Nicholls & Ferguson, 1997; Salway, 1999; Saraste,
1999). Ubiquinone, which accepts electrons and protons as it is reduced to
ubiquinol, shuttles fi-om both complexes I and II, to complex III. Similarly,
cytochrome c shuttles electons from complex III to complex IV.
33
-
Chapter 1
ATP ATP ATP
NADH+HMATRIX
FADH
...................... ADP-p.^mfumarate
ADP+P,—.««»« t-F^uprcinate
ATP synthetase
INNER
ÀMEMBRANE
INTERMEMBRANESPACE
Figure 1.7 The respiratory chain showing the flow of electrons from NADH
to oxygen and the extrusion of protons by complexes I, III and IV.
The synthesis of ATP is the result of two processes, electron transport and oxidative
phosphorylation and proton transport. Electron transport involves the oxidation from
NADH + H^, or FADH2 with transport of the electrons through the mitochondrial
respiratory chain until they are donated to molecular oxygen, which is subsequently
reduced to water. This electron transport, according to Mitchell’s chemiosmotic
theory, drives proton pumps in complexes I, III and IV by a mechanism still not fully
understood (Mitchell & Moyle, 1967). Positively charged protons, but not any
associated negatively charged anions, are pumped out of the mitochondrial matrix to
the cytosol (intermembrane space). Proton pumping is ultimately coupled to electron
flow so that there is no respiration without proton pumping and vice versa. As a
result, the matrix side of the membrane becomes negatively charged, whilst the
extruded protons ensure that its opposite side becomes positively charged thus
making the inner mitochondrial membrane anisotropic. The difference in
electrochemical potential across the membrane is about 150-250 mV (mitochondrial
34
-
Chapter 1
membrane potential) and it provides the energy for ATP synthesis when the protons
return to the matrix through the Fo proton channel and the Fi ATP synthase. At
present, it is the general consensus that three protons are needed to form one ATP
molecule and an additional proton is needed to translocate it to the cytosol. Key
membrane protein components of the mitochondrial respiratory enzymes and the
ATP synthase are encoded by genes in the mitochondrial DNA, and others are
encoded in the nucleus. The fundamental question of how ATP is synthesized by
FiFoATPase remains unanswered. Studies have led to the theory that a central
structure inside the FiATPase may be present that rotates, probably due to
protonmotive force, and could result in the translocation of four protons per one ATP
molecule synthesized (Sabbert et a l, 1996; Noji, 1998; Yasude et a l, 1998; Saraste,
1999). The determination of the three-dimensional structure of the entire ATP
synthase is necessary for a better understanding of the mechanism of ATP synthesis.
Mitochondria also take up calcium (Ca^^), thus contributing to the cellular
homeostasis, and are functionally tightly integrated into mechanisms of cellular
calcium signaling. Mitochondrial is regulated through trasport mechanisms, for
both Ca^ ̂ uptake and efflux, of the inner membrane (Gunter & Pfeiffer, 1990).
Uptake of Câ "̂ is through the potential-dependent uniporter, a mechanism driven by
the mitochondrial membrane potential. Three possible pumping mechanisms for the
efflux of Ca^ ̂have so far been identified: a 2 NaVCa^^ exchanger which is linked to
electron transport chain proton pumping via Na' /̂H' ̂ exchange; a sodium ion
independent mechanism, known to be an electroneutral Ca^V2H'*’ exchanger (Puskin
et a l, 1976; Haworth & Hunter, 1979; Bowser et a l, 1998) and a process known as
the mitochondrial transition pore (MTP) which may be due to a large proteinaceous
35
-
Chapter 1
pore (further discussed in chapter 5). The kinetics of mitochondrial Ca^ ̂uptake and
release appear to differ between cell types which may reflect differences in
intracellular Na^ availability for the mitochondrial NaVCa^ exchanger (Duchen,
1999).
Mitochondria are also important in glucose homeostasis. Mitochondrial respiration
is stimulated by the delivery of substrate in pancreatic y0 -cells and glucose-sensing
neurons of the hypothalamus which, results in the closing of certain channels in
the plasmalemma and the subsequent opening of voltage-gated Ca^^ channels. This
promotes insulin secretion, which in turn lowers plasma glucose (Duchen et ah,
1993; Duchen, 1999). Furthermore, the supply of glucose to the yg-cells, increases
substrate supply to the tricarboxylic acid cycle, increases the provision of NADH and
FADH2 to the respiratory chain, and increases respiratory rate which then leads to
increased ATP production.
36
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Chapter 1
1.5 Research objectives
As described in the previous sections enteroviruses have a tropism for muscle cells
and have been linked to the development of CFS and post-polio fatigue. Muscle
fatigue made worse by exercise is one of the major symptoms in CFS. Abnormalities
in cellular energy levels suggestive of an impairment of mitochondrial function have
been reported following virus infection. A mitochondrial disorder precipitated by a
virus infection has been suggested as the cause for the unexplained fatigue in CFS.
In addition, the PV non-structural proteins 2B and 2BC could possibly be involved in
the mitochondrial abnormalities since they have been extensively associated with
changes in membrane permeability and proliferation (as reviewed in sections 1 .2 . 6
and 1.3.2)
The main objective of this project was to establish whether mitochondrial function
was affected by PV infection and to identify the PV proteins involved in this effect.
This should lead to improved understanding of the muscle fatigue observed in
patients with CFS.
Specifically, mitochondrial function was investigated in different mammalian cell
lines infected with poliovirus, and the role of the non-structural poliovirus proteins
2B and 2BC was investigated:
1. To assess the effect of PV infection on cellular and mitochondrial respiration.
2 . If mitochondrial respiration was blocked to investigate the specific site of the
blockage.
3. To examine the mitochondrial membrane potential of PV infected cells.
37
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Chapter 1
4. To examine the consequences of PV infection on mitochondrial energy
production.
5. To express the proteins 2B and 2BC in mammalian in order to investigate
their possible effect on normal mitochondrial activity.
6. If time permits, raise antibodies specific for these proteins in order to
investigate their intracellular localisation.
38
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Chapter 2
Chapter 2
MATERIALS AND METHODS
39
-
Chapter 2
MATERIALS AND METHODS
2.1 Materials
2.1.1 Suppliers
Amersham International pic, Buckinghamshire, UK
dATP; conjugated anti-goat IgG-HRP antibodies.
Bio-Rad Laboratories Ltd, UK
Kaleidoscope pre-stained standards; acrylamide and bis-acrylamide; Whatman 3MM
filter paper.
Biotecx Laboratories, Inc, USA.
RNAzol B.
Boehringer Mannheim Ltd, Mannheim, Germany
CIP; lOx CIP buffer; restriction enzymes and their respective lOx buffers.
British Drug House (BDH) Ltd, UK
Ammonium acetate; butan-2-ol; liquid paraffin; acetone; isopropanol; polyethylene
glycol; ammonia.
Fischer Scientific International Co., UK
Ethyl alcohol; formaldehyde; methanol; potassium acetate; sodium hydroxide;
hydrochloric acid; acetic acid (glacial); formaldehyde; PCA; acetonitrile.
40
-
Chapter 2
Gibco-BRL, Paisley, Scotland
GMEM; RPMI-1640; DMEM; PCS; gentamycin; penicillin/streptomycin; versene;
trypsine; sodium bicarbonate; glutamine; formamide; lipofectin®; Opti-MEM® I
Reduced Serum Medium; PBS; electrophoresis grade agarose; RNAseIn;
bacteriophage lambda DNA; M-MLV RT.
Invitrogen
RNase ZAP, S.N.A.P.™ miniprep kit.
Millipore UK
Immobilon™-P nitrocellulose membrane.
Molecular Probes Europe BV
TMRE; Hoechst 33342.
National Diagnostics UK
HRPL substrates A and B.
Oxoid Ltd, Basingstoke, UK
Bacto agar; bacto tryptone; bacto yeast extract; nutrient broth; PBS tablets.
Pharmacia Biotech, UK
DEPC treated water; dNTPs; RNase H; pGEX-4T-2 and pGEX-4T-3 gene fusion
vectors; Glutathione Sepharose 4B beads; glutathione; goat anti-GST antibody.
41
-
Chapter 2
Promega Corp. UK
Wizard™ clean-up system; Klenow fragment; T4 DNA polymerase; lOx T4 DNA
polymerase buffer; T4 DNA ligase; lOx T4 DNA ligase buffer;.
QiagenLtd. UK
QIAquick gel extraction kit.
Sigma-Aldrich Chemical Co. Ltd, UK
Acrylamide; ammonium persulphate; ampicillin; BSA; kenacid blue; DTT; ethidium
bromide; IPTG; X-gal; TBMED; protein molecular weight markers; salmon sperm
DNA; 2-mercaptoethanol; DMSO; trypan blue dye; crystal violet; SDS; saponin;
NaCl; Hepes; ESIT; rotenone; succinic acid; SDS; malonate; chloroform; Tris-base;
EDTA; guanidine thiocyanate; MgCl2; CaCl2; KCl; boric acid; glycerol; MOPS;
polyvinyl pyrrolidone; frcoll; glycine; bromophenol blue; 0.4% (w/v) trypan blue;
ATP; Tween 20; potassium ferricyanide; potassium ferrocyanide; potassium
phosphate; ADP; AMP; adenosine; inosine.
42
-
Chapter 2
2.1.2 Solutions and buffers
2,1.2.1 Microbial growth medium
SOB medium
Bacto tryptone 20g
Bacto yeast extract 5g
NaCl 0.5g
250mMKCl 10ml
Distilled water to 11. The pH was adjusted to 7.0 with NaOH. After sterilisation by
autoclaving 5ml of sterile 2M MgCl] were added.
2xYT medium
Bacto tryptone 16g
Bacto yeast extract lOg
NaCl 5g
Distilled water to 11, sterilise by autoclaving.
2xYT medium used for inoculations contained ampicillin (50pg/ml).
Agar plates
1.2g of Bacto agar were added to 100ml 2xYT medium and boiled to dissolve. The
agar was then cooled to 50 ^C before adding ampicillin and if required X-Gal and
IPTG as follows: ampicillin stock solution (25mg/ml) was made in water and added
of a final concentration of 50pg/ml; 2% X-Gal stock solution was made in dimethyl
formamide and used at 1:200; O.IM IPTG was dissolved in water and used at 1:400.
43
-
Chapter 2
2.1.2.1 Buffers used in molecular cloning
5x first strand buffer
Tris-HCl, pH 8.3
KCl
MgClz
250mM
375mM
15mM
lOx second strand buffer
Tris-HCl, pH 8.3 188mM
KCl 906mM
MgCli 46mM
DTT 37.5mM
T4 DNA polymerase buffer
Tris-HCl, pH 7.9 0.33mM
Potassium acetate 0.66mM
MgClz O.IM
DTT 5mM
lOx ligation buffer
Tris-HCl, pH 7.6
MgCli
DTT
Spermidine
BSA
660mM
lOOmM
150mM
lOmM
2mg/ml
44
-
Chapter 2
2.1.2.3 Agarose gel DNA electrophoresis buffers
lOxTBE running buffer
Tris base 107.8g
Boric acid 55g
EDTA 9.3g
Distilled water to 11, sterilised by autoclave.
2xTBE sample buffer
lOxTBE running buffer 4ml
Glycerol 4ml
Distilled water 12ml
Bromophenol blue and xylene cyanol FF dyes were added to this buffer until its
colour became an intense purple/blue.
2.1.2.4 Northern blotting solutions
lOxMOPS buffer
MOPS 0.2M
Sodium acetate 0.05M
EDTA O.OIM
pH 7.0 with NaOH
Sample buffer
Formamide 0.5ml
lOxMOPS 0.1ml
Formaldehyde 0.15ml
45
-
Chapter 2
Pre-hybridisation solution
20xSSC 15ml
200mM EDTA 25ml
lOOx Denhadt’s solution 2.5ml
20% SDS 1.25ml
Carrier DNA 0.5ml
Distilled water 28.25ml
Pre-hybridisation solution was stored at 4 ®C, and warmed to re-dissolve.
lOOx Denhardt’s solution- 5 g bSA; 5g polyvinyl pyrrolidone; 5g ficoll; water to
250ml.
Carrier DNA: lOmg/ml salmon sperm DNA in distilled water, boiled and sheared.
Hybridisation wash solution: 2 % SSC plus 0.1% SDS.
OLB solution: 250mM Tris-HCl, pH8.5; 25mM 2-mercaptoethanol; OlmM each of
dCTP, dGTP, dTTP; IM Hepes, pH 6.6.
Stop/quench solution:
4M NaCl 50 pi
IM Tris, pH 7.5 200pl
200mM EDTA lOOpl
20% SDS 125pl
Distilled water 9.50pl
46
-
Chapter 2
2.1.2,5 SDS-PAGE solutions
Resolving buffer: 0.75M Tris, pH 8.8 with concentrated HCl
Stacking buffer: 0.25M Tris, pH 6.8 with concentrated HCl
Acrylamide:bis-acrylamide: 4 q% stock by mixing 40g acrylamide with 1.08g bis-
acrylamide and made to 100ml with distilled water.
Running buffer
Tris 3.02g
Glycine 14.44g
20% SDS 5ml
Distilled water to 11, pH 8.2
Ix loading dye
Stacking buffer 2.5ml
20% SDS 1ml
Glycerol 2g
2-mercaptoethanol 0.25ml
1% bromophenol blue 0.05ml
Distilled water to 10 ml.
47
-
Chapter 2
10% resolving gel
Distilled water 4.6ml
Resolving buffer 10ml
Acrylamiderbis-acrylamide 3.8ml
20% SDS 0.1ml
Ammonium persulfate 0.6ml
TEMED 12pl
4% stacking gel
water 3.75ml
Stacking buffer 5ml
Acrylamide:bis-acrylamide 1ml
20% SDS 50pl
Ammonium persulfate 0.25ml
TEMED lOpl
48
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Chapter 2
2.2 Mammalian cell culture
2.2.1 Cell lines
Three mammalian cell lines were used during the course of this study:
Vero cells
African green monkey kidney anchorage-dependent fibroblasts that grow
continuously in culture. These cells are susceptible to a wide range of viruses
including poliovirus and reoviruses.
The cells were grown in Glasgow’s modified Eagles basal medium (GMEM),
supplemented with 10% (v/v) heat inactivated foetal calf serum (ECS), 2mM
glutamine, 1% (v/v) gentamycin, 1% (v/v) penicillin/streptomycin and 2.7% (v/v)
sodium bicarbonate, pH 7.0. Incubation of the cells was carried out at 37 °C in a
humidified atmosphere of 5% CO2 in air. Once the cells formed a confluent
monolayer, they were subcultured in the following manner: the medium was
aspirated and the cells were detached by the addition of versene containing 10% (v/v)
trypsin. Pre-warmed (37 °C) GMEM was added to the detached cells and they were
centrifuged for 5 min at 1,500 rpm. The supernatant was then discarded and the cells
were split 1:3 in fresh GMEM and added to new tissue culture flasks containing an
appropriate volume of fresh GMEM.
COS-1 cells
Afirican green monkey kidney CV-1 cells (fibroblasts) transformed with SV40 DNA.
These also grow continuously in culture and they are possible hosts for the
propagation of recombinant SV40 virus.
49
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Chapter 2
The cells were grown in DMEM, supplemented with 10% (v/v) heat inactivated FCS,
2mM glutamine, 5% (v/v) gentamycin, 1% (v/v) penicillin/streptomycin and 5%
(v/v) sodium bicarbonate, pH 7.0. Incubation of the cells was carried out at 37 °C in
a humidified atmosphere of 5% CO2 in air. Once the cells formed a confluent
monolayer they were subcultured in the same manner used for Vero cells.
T47D cell
A continuous human breast carcinoma epithelial cell line carrying receptors to a
variety of steroids.
The cells were grown in RPMI 1640 medium; supplemented with 10% (v/v) heat
inactivated FCS, 2mM glutamine, 1% (v/v) gentamycin, 1% (v/v)
penicillin/streptomycin and 2.7% (v/v) sodium bicarbonate, pH 7.0. Incubation of the
cells was carried out at 37 °C in a humidified atmosphere of 5% CO2 in air. Cell
growth was slow and it required addition of fresh media for the cells to reach
confluency. Once the cells formed a confluent monolayer they were subcultured in
the same manner used for Vero cells.
2.2.2 Freezing of cell lines
Confluent cells were detached using versene/trypsin and centrifuged as described in
section 2.1.1. The cells were then resuspended in sufficient, pre-warmed (37 °C),
medium (80% (v/v) normal growth medium, 10% (v/v) heat inactivated FCS, 10%
(v/v) dimethyl sulphoxide (DMSO)) to give a final cell density of 4-6 x 10 ̂cells/ml.
50
-
Chapter 2
1 ml aliquots of the resuspended cells were pipetted into cryotubes and frozen slowly
to -70 °C overnight before storage in liquid nitrogen.
Cells were recovered after storage, by thawing a frozen aliquot was thawed rapidly in
a 37 °C waterbath. The cell suspension was then diluted with 10 ml of the respective
growth medium (pre-warmed to 37° C) and centrifuged at 1,500 rpm for 5 min to
remove the DMSO. The cell pellet was resuspended in 5 ml of growth medium,
transferred to a small tissue culture flask and incubated at 37° C in a humidified
atmosphere of 5% CO2 in air. The medium was changed 24hrs later to ensure the
complete removal of the DMSO. The cells were then subcultured as described in
section 2.1.1.
2.2.3 Cell counting
Cell density was determined by adding lOOpl 0.4% trypan blue dye (w/v) in PBS to a
lOOpl cell suspension. Counting was carried out by introducing a sample of the
stained cell suspension into a Neuba